Chemistry

The Royal Swedish Academy of Sciences has awarded the 1997 Nobel Prize in Chemistry with one half to Paul D. Boyer (University of California, Los Angeles, USA) and John E. Walker (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) for elucidation of the mechanism of action of ATP synthase, which catalyzes the synthesis of adenosine triphosphate (ATP); and one half to Jens C. Skou (Aarhus University, Denmark) for the first discovery of an ion-transporting enzyme, Na⁺,K⁺-ATPase. The three laureates have performed pioneering work on enzymes that catalyze reactions of the "high-energy" compound adenosine triphosphate (ATP).

ATP: The Universal Energy Carrier in the Living Cell

The German chemist Karl Lohmann discovered ATP in 1929. Its structure was determined some years later; and in 1948, the English Nobel laureate of 1957, Alexander Todd, synthesized ATP chemically. During the years 1939­1941 the 1953 Nobel laureate in Medicine, Fritz Lipmann, showed that ATP is the universal carrier of chemical energy in cells in all living organisms, from bacteria and fungi to plants and animals (including humans). ATP has been termed the cell's energy currency.

Adenosine triphosphate (ATP) consists of adenosine linked to three phosphate groups. On removal of the outermost phosphate group, adenosine diphosphate (ADP) is formed. The energy released can cause other reactions to occur. Conversely, with the help of energy, an inorganic phosphate group can be bound to ADP to form ATP. In one day an adult human at rest converts a quantity of ATP corresponding to about one-half of the body's weight. During hard work the quantity can rise to almost a ton. Most of the ATP synthesis is carried out by the enzyme ATP synthase. At rest, Na⁺,K⁺-ATPase uses up one third of all ATP formed.

ATP Synthase: An Exceptional Molecular Machine

During the 1940s and 1950s it was determined that the bulk of ATP is formed during cell respiration in the mitochondria and photosynthesis in the chloroplasts of plants. In 1960 the American scientist Efraim Racker and his coworkers isolated from mitochondria the enzyme F_oF₁ATPase, which we now call ATP synthase. The enzyme can be divided into an F₁ part containing the catalytic center and an F_o part, which couples the F₁ part to the membrane (see Fig. 1). In 1961, Peter Mitchell, who received the Nobel prize in 1978, showed that cell respiration leads to a difference in pH inside and outside the mitochondrial membrane, and that a stream of hydrogen ions drives the formation of ATP. The coupling of ATP synthase to hydrogen ion transport takes place via the F_o part.

Paul D. Boyer began his studies of ATP formation in the early 1950s, seeking to learn by isotope techniques how ATP synthase functions and particularly how it uses energy to create new ATP. His work has been crowned with unusual success in the past few years. ATP synthase has a mode of function unusual for enzymes, and this required much time and extensive studies to establish. John E. Walker made his first studies of ATP synthase at the beginning of the 1980s. His starting thesis was that a detailed chemical and structural knowledge of an enzyme is required to understand in detail how it functions. He therefore determined the amino acid sequences in the constituent protein units. During the 1990s he has collaborated with crystallographers to elucidate the three-dimensional structure of ATP synthase. So far, the structure of the enzyme's F₁ part has been established. Walker's work complements Boyer's in a remarkable manner and further studies based on this structure demonstrate the correctness of the mechanism proposed by Boyer.

ATP synthase (Fig. 1) consists of a membrane-bound part, F_o , which transports hydrogen ions, and a protruding part (F₁), which carries out its catalytic function. Each F_o part consists of three types of protein subunits in differing numbers: a (1), b (2), and c (9-12). The F₁ part consists of five subunits, alpha, beta, gamma, delta, and epsilon. There are three each of alpha and beta subunits, but only one unit of each of the others. It has been shown that the synthesis of ATP occurs on the beta units. The analysis of amino acid sequences that Walker and coworkers did at the beginning of the 1980s showed that subunits gamma, delta, and epsilon are not symmetrical - a feature important for understanding how ATP synthase functions.

Boyer and coworkers found that despite the asymmetrical character of F₁, there is only one way for the enzyme to react. But how then can the three beta subunits function in the same way if they have different couplings to subunits gamma, delta, and epsilon? Boyer suggested that gamma, delta, and epsilon rotate in a cylinder formed of alternating alpha and beta subunits. This rotation induces structural changes in beta, which lead to differences in bonding ability during a cyclical course (see Fig. 2). This is called Boyer's "binding change mechanism". Boyer also proposed that this rotation is driven by hydrogen ion flow through the membrane.

Figure 2 shows the cylinder with alternating alpha and beta subunits at four stages of ATP synthesis. The asymmetrical gamma subunit that causes changes in the structure of the beta subunits can be seen in the center. The structures are termed open beta_o (light grey sector), loose beta_L (grey sector) and dense beta_T (black sector). At stage A we see an already-fully-formed ATP molecule bound to beta_T. In the step to stage B, beta_L binds ADP and inorganic phosphate (P_i). At the next stage, C, the gamma subunit has twisted owing to the flow of hydrogen ions (see Fig. 1). This brings about changes in the structure of the three beta subunits. The dense b subunit now becomes loose and the bound ATP molecule is released. The loose beta subunit becomes dense and the open becomes loose. In the last stage, the chemical reaction takes place in which phosphate ions react with the ADP molecule to form a new ATP molecule. We are back at the first stage.

Boyer has called ATP synthase a molecular machine. It may be compared to a water-driven hammer minting coins. The F_o part is the wheel, the flow of protons is the waterfall, and the structural changes in F₁ lead to three coins in the ATP currency being minted for each complete turn of the wheel.

Na⁺,K⁺-ATPase, First Molecular Pump To Be Discovered

It was known as early as the 1920s that the ion composition within living cells is different from that in the surroundings. Within the cells the sodium concentration is lower and the potassium concentration higher than in the liquid outside. Through the work of the Englishmen Richard Keynes and Alan Hodgkins at the beginning of the 1950s (Hodgkins received the Nobel prize in 1963), it was known that when a nerve is stimulated sodium ions pour into the nerve cell. The difference in concentration is restored by sodium being transported out once again. That this transport required ATP was probable, since the transport could be inhibited in living cells by inhibiting the formation of ATP.

With this as the starting point, Jens C. Skou searched for an ATP-degrading enzyme in the nerve membrane that could be associated with ion transport. In 1957 he published the first article on an ATPase, which was activated by sodium and potassium ions (Na⁺,K⁺-ATPase). He was the first to describe an enzyme that can promote directed (vectored) transport of substances through a cell membrane, a fundamental property of all living cells. Since more sodium ions are transported out than potassium ions are transported in, an electrical potential is created across the membrane.

This difference in potential across the membrane is a condition for a nerve stimulation to propagate along a nerve fiber or a muscle cell. This is why a shortage of nourishment or oxygen in the brain rapidly leads to unconsciousness. ATP formation ceases, there is no ATP for ATPase to act on, and the ion pump stops. The pump is also important for maintaining cell volume. If the pump stops, the cell swells. The difference in sodium-ion concentration between the interior and the exterior is the driving force in the uptake of nutrients necessary to the cell, such as glucose and amino acids. It can also be used for transport of other ions through the cell membrane. Thus sodium ions that enter can be exchanged for calcium ions that exit. The latter is the mechanism that enables digitalis to strengthen heart activity.

Medicine

The Nobel Assembly at the Karolinska Institute has awarded the Nobel Prize in Physiology or Medicine for 1997 to Stanley B. Prusiner for his discovery of prions - a new biological principle of infection. [See "Sick Cows, Protein Geometry, and Politics", J. Chem. Educ. 1996, 73, A232­A233.]

Stanley Prusiner has added prions to the list of well-known infectious agents including bacteria, viruses, fungi, and parasites. Prions exist normally as innocuous cellular proteins. However, prions possess an innate capacity to convert their structures into highly stable conformations that ultimately result in the formation of harmful particles, the causative agents of several deadly brain diseases of the dementia type in humans and animals. Prion diseases may be inherited, be laterally transmitted, or occur spontaneously. Regions within diseased brains have a characteristic porous and spongy appearance, evidence of extensive nerve cell death, and affected individuals exhibit neurological symptoms including impaired muscle control, loss of mental acuity, memory loss, and insomnia. Stanley Prusiner's discovery provides important insights that may furnish the basis to understand the biological mechanisms underlying other types of dementia-related diseases, for example Alzheimer's disease, and establishes a foundation for drug development and new types of medical treatment strategies.

The Prize-winning Research Initiated 25 Years Ago

In 1972 Stanley Prusiner began his work after one of his patients died of dementia resulting from Creutzfeldt­Jakob disease (CJD). It had already been shown that CJD, kuru, and scrapie, a similar disease affecting sheep, could be transmitted through extracts of diseased brains. There were many theories regarding the nature of the infectious agent, including one that postulated that the infectious agent lacked nucleic acid - a sensational hypothesis, since at the time all known infectious agents contained the hereditary material DNA or RNA. Prusiner took up the challenge to precisely identify the infectious agent; and ten years later, in 1982, he and his colleagues successfully produced a preparation derived from diseased hamster brains that contained a single infectious agent. All experimental evidence indicated that this agent comprised a single protein, a "proteinaceous infectious particle" which Prusiner named a prion, to distinguish it from a virus or a virioid. The scientific community greeted this discovery with great skepticism. However, an unwavering Prusiner continued the arduous task to define the precise nature of this novel infectious agent.

No Intrinsic Defense Mechanisms against Prions

Prions are much smaller than viruses. The immune response does not react to prions because they are natural proteins, present from birth. They become deleterious only by converting into a structure that enables disease-causing prion proteins to interact with one another forming threadlike structures and aggregates that ultimately destroy nerve cells. The mechanistic basis underlying prion protein aggregation and the cumulative destructive mechanism are still not well understood. The ability to transmit a prion infection from one species to another varies considerably and is dependent upon what is known as a species barrier. This barrier reflects the nature and extent of the structural relationship between prions of different species.

The information about the 1997 Nobel Prizes was adapted from the press releases of the Royal Swedish Academy of Sciences. Further information is available from the Academy of Sciences, Information Department, Box 50005, SE-104 05 Stockholm, Sweden. Phone: +46 8 673 95 25; fax: +46 8 15 56 70; email: info@kva.se; Web site:www.kva.se.